Figure 3.

Development protocol for spheroids and organoids

Both models can be obtained from different sources (cyan panel) -immortalized cell lines, embryonic stem cells (ESC), pluripotent stem cells (PSC), NAFLD murine models or liver from NAFLD patients. Immortalized cell lines and differentiated ESC/PSC self-aggregate to form spheroids. To induce the NAFLD phenotype, free fatty acids (FFA) supplementation is required. To form liver organoids, cells from liver specimens are extracted and isolated. Hence, they are exposed to selected growth factors in presence of a basement matrix. These liver organoids then differentiate into the specific cell lines and acquire the NAFLD phenotype upon FFA supplementation (light green panel). In addition, differentiated organoids can also be obtained from ESC/PSC or immortalized cell lines aggregated in liver spheroids. After dissociation of the aggregates and subsequent cultivation with a basement matrix, the differentiated organoids are ready to be used for NAFLD studies. The heterogeneity of the disease, its complexity, the self-organization of the cells or the life-span of the cultures is higher in organoids than in spheroids.